Abstract: This project will use the analysis of pluripotent stem cells and pre-implantation embryonic development as testbed questions to build and test quantitative evolutionary models for gene regulatory networks (GRNs). Pluripotency refers to the potential of an undifferentiated cell to differentiate into any of the three germ layers and give rise to any adult cell type. The general hypothesis of the project is that the pluripotent cell phenotype can be sustained by alternatively implemented (re-wired) GRNs across species. This hypothesis deduces that the knowledge of the re-wiring of GRNs will contribute to finding new routes and efficient methods for reprogramming somatic cells into a pluripotent state. This hypothesis will be addressed by developing methods for combining multi-modality data for GRN reconstruction and quantitative phylogenetic models for GRN evolution. These models will be applicable for identifying and analyzing GRNs in other species and other biological processes. This project will build a general probabilistic framework to enable joint analysis of genomic, epigenomic and transcriptomic data as well as inference of combinatorial and evolutionary rules that GRNs conform to and implement. Five research thrusts will be developed as follows. 1. Develop a probabilistic framework for GRN analysis. 2. Develop models for combinatorial interactions of TFs with a cis-regulatory module (CRM), and extend this model to incorporate epigenetic states of the CRM. 3. Develop probabilistic evolution models for identification of conserved and species- specific gene expression modules. 4. Develop an evolutionary model for analysis of re-wiring of GRNs. The co-evolution of the regulatory relationships of TF and target genes, CRMs and the expression levels of target genes will be modeled. 5. Identify the conserved and re-wired components of the GRNs that support the pluripotent cell identity mammals. Experimentally test these conserved and species-specific regulatory relationships in human and mouse embryonic stem cells. Public Health Relevance: This project will use the analysis of pluripotent stem cells and pre-implantation embryonic development as testbed questions to build and test quantitative evolutionary models for gene regulatory networks. This project will address how gene expression is regulated in pluripotent stem cells and how such gene regulatory networks evolve. Such information may lead to finding new and more efficient routes of cellular reprogramming, which is critical to the development of cell-based therapies for various diseases.